As a strategy to identify new virulence-associated factors, we are focusing our attention on proteins that exported by Legionella and proteins that are localized in the bacterial envelope. This approach is based on the assumption that bacterial products which have a role in critical host-parasite interactions are likely to be surface-expressed or exported. We can specifically identify mutations in genes that encode secreted proteins by generating translational fusions to the gene that encodes the alkaline phosphatase of E. coli, since this enzyme is expressed only when it is localized exterior to the cell membrane. Using two different techniques, we have constructed several phosphatase-positive L. pneumophila mutants that have attenuated virulence for human macrophages. We are presently characterizing these mutants functionally and sequencing the targeted genes to determine their identities.